Conventionally, in the field of genetic engineering, in order to perform DNA cloning, gene therapy, or breeding, a vector is necessary as a carrier which brings a foreign target gene into a cell of an organism, in addition to enzymes which cut and paste genes. If a bacterium such as E. coli as a host is used as such a vector, a plasmid or a λ phage has been used and mixed with E. coli and the like, then electroshocked to introduce this. Moreover, in order to separate the E. colis introduced in this manner, the process has been such that, after a series of treatments to combine an antibiotic resistant gene resistant to antibiotics into the plasmid or the like, only cells which are transformed through the antibiotic treatment are extracted.
Moreover, there have been three methods for bringing a foreign target gene into an animal cell namely; (1) to introduce a foreign gene as is, (2) to introduce it by microinjection, and (3) to make a retrovirus carry it.
Introducing a foreign target gene as is, into an animal cell is performed by mixing calcium phosphate with the foreign genes, and then mixing the precipitated genes with the animal cells. The reason for this is that by so doing even a small number of cells can take-in the precipitated genes.
In the microinjection, a foreign gene is directly inserted into a cell nucleus using a micropipette (extra fine capillary tube) which is made from very fine glass having a diameter of 0.1 μm, while watching through a microscope.
Moreover, in order to use a retrovirus as a vector, a foreign target gene is sandwiched between the opposite terminals by a retrovirus LTR (transcriptional promoter and poly A-binding site) to create the vector, which is then introduced into an animal cell.
Furthermore, there is another method wherein the biological material is introduced using magnetic particles by shooting the magnetic particles fixed with the biological materials into cells, organs, or tissues at high speed (initial velocity is 50 to 400 m/second) (for example, refer to Japanese Unexamined Patent Publication No. 6-133784, paras. 14,19, and 20). This method is to facilitate the concentration or separation of cells by using magnetic force.
Incidentally, in the conventional method of introducing a target gene described above, a parasitic genetic factor (extrachromosomal gene of a bacterium which can proliferate independently from chromosomes and the like) such as a plasmid or a virus such as a λ phage has been used as a vector. There has been a problem in that, the parasitic genetic factor and the virus are intrinsically pathogenic in many cases, and even if the pathogenicity is put out of action in use, they may recover the pathogenicity by recombining with another pathogen infected to the host. Moreover, there has been another problem in that, even if electroshock is applied during the introduction, the introduction efficiency is not high enough. Furthermore there has been another problem in that, due to the presence of the antibiotic resistant gene used when the introduced cells are extracted, the diffusion of the antibiotic resistant gene may negatively affect the environment of the host.
If the host is an animal cell, the first method can be readily performed since the operating procedure is simple. However, this method has a problem of extremely poor efficiency. The second method allows introducing the target gene into the cell nucleus reliably. However, there has been a problem in that to master this technique requires a reasonable technical training and takes time.
In the third method, a package cell strain having the autonomous replication-competence deleted and genes required for proliferation combined into a chromosome is used for ensuring the safety of the retrovirus. However, there has been a problem in that, similarly to the above mentioned plasmid, the retrovirus may recover the pathogenicity by recombining with another pathogen infected to the host.
In the method of shooting the magnetic parties fixed with the biological materials into cells at high speed using a firing apparatus, there have been problems in that: the scale of the apparatus becomes larger; and that the magnetic particles which are once fired and go astray from the cells can not be reused, and hence the efficacy is low after all. There has been another problem in that, since they are shot at high speed, cells having low strength are broken down when the parties collide with the cells, making it impossible to introduce the biological materials thereinto.
As described above, any method has a problem of poor efficacy, since the way to introduce a biological material into a host is to let the host and the biological material suspended or contained in the solution encounter naturally, or to introduce the biological material into the host one by one manually, or only once by a firing apparatus.
Therefore, the present invention has been developed to solve the above mentioned problems, with a first object of providing an apparatus for introducing a biological material, a method of introducing a biological material and a magnetic support for introducing a biological material, whereby a foreign biological material can be introduced into a host efficiently, not by relying on a natural encounter of a host and a biological material but by using a magnetic force and controlling its magnitude, direction, location, and the like, to promote the collision by moving the magnetic support until it enters the host, or to promote the encounter by densifying the biological material in the solution.
A second object is to provide an apparatus for introducing a biological material a method of introducing a biological material and a magnetic support for introducing a biological material wherein various treatments are standardized and automatically performed without relying on manpower nor skilled technicians, so that the introduction into a host can be readily performed.
A third object is to provide an apparatus for introducing a biological material a method of introducing a biological material, and a magnetic support for introducing a biological material whereby the introduction can be performed without using a recombinant of a virus or a parasitic genetic factor such as a plasmid, and a biological material can be introduced safely.
A fourth object is to provide an apparatus for introducing a biological material, a method of introducing a biological material, and a magnetic support for introducing a biological material whereby a host having a foreign biological material introduced thereinto can be readily and reliably separated and extracted.
A fifth object is to provide an apparatus for introducing a biological material a method of introducing a biological material and a magnetic support for introducing a biological material, whereby the effect on the environment caused by combining an antibiotic resistant gene can be avoided, enabling safe separation and extraction.
A silt object is to provide an apparatus for introducing a biological material, a method of introducing a biological material, and a magnetic support for introducing a biological material which has variety and generality allowing introduction under various conditions according to the properties of hosts and biological materials.